Simultaneous sequencing of nucleic acids

ABSTRACT

The invention concerns a new method for the simultaneous sequencing of nucleic acids using numerous double-stranded DNA adaptors.

This application is a National Stage Application of PCT Application No.PCT/EP93/01376, filed Jun. 1, 1993, which claims priority from GermanApplication No. P 42 18152.6, filed Jun. 2, 1992.

The present invention concerns a new method for the simultaneoussequencing of nucleic acids as well as a method for converting analready existing gene bank into a modified gene bank on which asimultaneous sequencing of nucleic acids can be carried out.

The sequence of nucleic acids is usually determined either by chemicalDNA sequencing (Maxam-Gilbert technique) or by the enzymatic chaintermination method (Sanger technique). In the Maxam-Gilbert technique abase-specific cleavage of the nucleic acid to be sequenced is carriedout with the aid of certain chemicals. In the Sanger technique anenzymatic polymerization reaction is carried out using the nucleic acidto be sequenced as a template and a DNA polymerase, e.g. the Klenowfragment of E. coli DNA polymerase, of T4 DNA polymerase or of T7 DNApolymerase. 2',3'-dideoxynucleoside-5'-triphosphates (ddNTP) are used asa substrate for the enzyme as chain terminating molecules in addition tothe normal deoxynucleoside triphosphates (dNTP). Incorporation of thesedideoxy compounds into a freshly synthesized DNA strand causes atermination of the polymerization reaction.

The sequence of the individual steps in the procedure is essentially thesame in both of the aforementioned techniques: Firstly a high molecularDNA to be sequenced is broken down into several smaller fragments whosesequence can then be determined. This fragmentation can for example beachieved by specific cleavage with restriction enzymes or also byunspecific cleavage with DNase I or by ultrasonic treatment of the DNA.

The actual determination of the sequence is carried out on the smallerfragments, the principle of both sequencing procedures being that apopulation of sequencing products of different length whose nucleotidesequence derives from that of the DNA to be sequenced is generated by achemical or enzymatic reaction. One end of these sequencing products isidentical for the entire population and the other end is a variable endwith one in each case of the four possible bases of DNA. The sequencingproducts of different length are then separated from one another byseparation according to size in general by gel electrophoresis in verythin, high resolution denaturing polyacrylamide gels.

The determination of the sequence is generally carried out by directanalysis of these sequence gels. In this case the sequencing productsmust have a direct label e.g. by incorporation of radioisotopes such as³² P or ³⁵ S or biotinylated or fluorescent-labelled nucleotides.

The disadvantage of a direct labelling of the sequencing products isthat always only one single DNA fragment can be processed in each casein each base-specific sequencing reaction. This disadvantage can beeliminated by a simultaneous sequencing of nucleic acids according tothe so-called multiplex technique (see e.g. EP-A 0 303 459). Thistechnique allows the simultaneous processing of several, in general upto 50 different DNA fragments in each base-specific sequencing reaction.For this the DNA fragments to be sequenced are incorporated intodifferent cloning vectors. These vectors differ in each case in thatoligonucleotide sequences that are specific for the vector used arelocated to the left and right of the cloning site. All vectors carry thesame cleavage site for a restriction enzyme outside theseoligonucleotide sequences with which the cloned DNA fragments togetherwith the neighbouring regions which are specific for them can be againcleaved from the vector. A Maxam-Gilbert sequence reaction is carriedout on this fragment mixture. After gel-electrophoretic separation ofthe sequencing products, the bands are transferred from the sequence gelonto a nylon membrane and are immobilized there. By hybridizing thismembrane with hybridization probes that in each case are specific foronly one vector it is possible to visualize the sequences of several DNAfragments in succession on the same membrane. The advantage of thismethod is that the four base-specific sequencing reactions and theseparation of the sequencing products which are obtained thereby onlyhave to be carried out once.

However, a disadvantage of this strategy is that numerous vectors eachwith different specific oligonucleotide sequences are necessary to clonethe DNA fragment. A further disadvantage of the multiplex procedure isthat the cloning of the DNA into the individual vectors is solelypossible by a blunt end ligation. Not only is the cloning efficiencycomparably low in this type of cloning but a certain percentage of thecolonies contain pure vector DNA due to the low ligation efficiency.This portion has to be separated by a preparative gel electrophoresisbefore the sequencing.

As a result of the aforementioned disadvantages the multiplex sequencingstrategy only has a relatively limited application. Thus the object ofthe present invention was to develop a method for the simultaneoussequencing of nucleic acids in which the aforementioned disadvantages,in particular the use of numerous base vectors and the low cloningefficiency is eliminated.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the components required as starting materials for apreferred embodiment of the method according to the invention, a vector(A) and two adaptor molecules (B) and (C).

FIG. 2 shows the hydrolysis of the vector (A) with restriction enzymes.

FIG. 3 illustrates the ligation of sequencing DNA (D) to the adaptormolecules (B) and (C), and the cloning of this complex into the openedvector (A).

FIG. 4 shows schematically a particularly preferred embodiment forconverting conventional gene banks into gene banks on which asimultaneous DNA sequencing is possible.

The object according to the invention is achieved by a method for thesimultaneous sequencing of nucleic acids which is characterized by

(a) provision of numerous DNA fragments whose sequence is to bedetermined,

(b) division of the totality of the DNA fragments to be sequenced intoseveral groups,

(c) ligation of double-stranded DNA adaptors to both ends of the dividedDNA fragments from (b) wherein the adaptors have a double-strandedregion with a length of ≧5 nucleotides, have a first end that iscompatible to the ends of the DNA fragments and a second end suitablefor cloning into a vector and wherein adaptors having a differentnucleotide sequence are ligated to each group of the divided DNAfragments,

(d) cloning the DNA fragments from (c) that are provided at both endswith adaptors into a base vector which has restriction sites matchingthe second ends of the adaptors,

(e) selection of a number of clones from (d), in which however, only 1clone at most may originate from one group and carrying out sequencingreactions on the selected clones and

(f) analysis of the results of the sequencing reactions using a numberof detectable hybridization probes each of which are specific for onlyone single clone from (e).

The advantages of the method according to the invention compared to theknown multiplex method are in particular that, in contrast to the knownmultiplex system, only a single base vector is required. As aconsequence the adaptor sequences which in the last reaction stepdetermine the binding of the hybridization probe used in each case tothe immobilized nucleic acid, can already be ligated to the DNAfragments to be sequenced outside the vector. Thus the totality of theDNA fragments to be sequenced can be divided into any desired number ofgroups since the number of these groups is not limited by the number ofavailable vectors. The required different adaptor oligonucleotides whichare preferably 10 to 40 nucleotides long can be chemically synthesizedin a simple manner with any desired sequences. The efficiency of theligation of the DNA fragments with the adaptor molecules can beconsiderably improved by using an excess of adaptor molecules. This alsoresults in higher yields of colonies obtained per DNA used in comparisonto the known multiplex system.

The DNA fragments provided according to step (a) of the method accordingto the invention can have blunt or stepped ends. DNA fragments withblunt ends can for example be obtained by cleaving the DNA and ifnecessary subsequent polishing of the ends in any desired manner. TheseDNA fragments are preferably produced by disintegrating DNA by means ofultrasound and enzymatic treatment (e.g. with T4 DNA polymerase orKlenow polymerase) of the ends, cleavage with blunt-cutting restrictionenzymes, mechanical shearing of DNA or/and by DNase treatment. DNAfragments with stepped ends are preferably produced by cleaving DNA withappropriate restriction enzymes or/and other sequence-specific cuttingenzymes.

Step (b) of the method according to the invention comprises dividing thetotality of the DNA fragments to be sequenced into several groups. Therecan be any desired number of these groups and it depends for example onthe size of the total DNA to be sequenced. For example the DNA fragmentsto be sequenced can be divided into 10 to 1000, preferably 50 to 500groups or fractions.

These individual groups of DNA fragments are ligated according to step(c) of the method according to the invention to double-stranded DNAadaptors which on one side have a first end that is compatible to theDNA fragments. This end can be blunt or stepped as set forth above. Theadaptors have a second end on their other side which is suitable forcloning into a vector. This end can be blunt but it is preferablystepped since in this case the cloning of the DNA fragments to besequenced into the base vector is not carried out via a blunt endligation but rather via a ligation of DNA fragments with stepped ends sothat a substantially improved cloning efficiency is found.

It is expedient that the length of the double-stranded region of anadaptor is sufficient to enable a ligation of the adaptor to the DNAfragment. In general the double-stranded region must therefore be ≧5nucleotides, preferably ≧6 nucleotides long.

Adaptors of a different nucleotide sequence are ligated to each group ofthe divided DNA fragments. In this process within one group one caneither use a single adaptor or even a mixture of two adaptors whichdiffer in their nucleotide sequence or/and in their second end (intendedfor cloning into the vector). In this connection the term "differentnucleotide sequence" means that the nucleotide sequence of adaptors fromdifferent groups (or even within a group) differs to the extent that nocross-hybridization takes place during the analysis of the sequencingreaction using different labelled hybridization probes in step (f) ofthe method according to the invention. Further criteria which shouldpreferably be observed when selecting adaptor sequences are a meltingpoint that should be as high as possible (GC content), the absence ofinternal hairpin structures and the presence of "rare" sequences whichonly occur with a low probability in the DNA fragments to be sequenced(e.g. 5'-CG-3' in eukaryotic DNA). In addition it is preferred that theadaptor sequences used have a melting point which is as uniform aspossible so that the sequencing can be more readily automated.

For the ligation reaction within each group one can use a single adaptoror two adaptors with a different nucleotide sequence or/and differentsecond ends, the latter possibility being preferred. If differentadaptors are used with different second ends it is therefore necessaryto use a base vector for the cloning of the DNA fragments provided withadaptors which has been opened at two different restriction sitesmatching the respective ends of the adaptors, i.e. has beenunsymmetrically hydrolysed. The restriction sites for cloning the DNAfragment are arbitrary provided that when they are cleaved with therespective enzyme they yield matching (and preferably non-compatible)ends. Examples of this are EcoRI, PstI, HindIII sites etc. In additionit is of course expedient that the restriction sites used for thecloning are singular sites on the base vector.

The base vector preferably has at least one so-called "rarely occurring"enzymatic cleavage site on both sides of the insertion site. A rarelyoccurring cleavage site is understood as a cleavage site, which due toits specific characteristics, is expected to occur within the DNA to besequenced only with a very low probability. Examples for such cleavagesites are for instance restriction sites with a recognition sequence ofat least 7 nucleotides (e.g. NotI, SfiI, RsrII, SgrAI, SwaI, PacI, AscI,PmeI, Sse8387I, SrsI or I-SceI sites) or/and those restriction siteswhich contain the nucleotide sequence 5'-CG-3' at least once withintheir recognition sequence which occurs extremely rarely in eukaryoticDNA. This rarely occurring cleavage site can be used to cut out thecloned DNA from the vector without a cleavage within the DNA.

In addition it is preferred but not necessary to use a base vector forthe cloning of the DNA fragments provided with adaptors which hastranscription terminators in the vicinity of the restriction sites usedfor the insertion of the DNA fragments so that no negative interactionsof the vector with the host cell due to a possible transcription of thecloned DNA sequences can occur. Preferred examples of suitable basevectors are for instance those which have a "multiple cloning site" withcorresponding suitable singular restriction sites for cloning. Examplesof this are in particular the commercially available dideoxy sequencingvectors which are set forth in Table 7.1.1 of Ausubel et al., CurrentProtocols in Molecular Biology, Supplement 16 (chapter 7.1.3). Fromthese statements it is clear that the requirements for the usability ofa vector for the method according to the invention are much less thanfor the usability of a vector in the known multiplex method.

Step (e) of the method according to the invention comprises theselection of a number of different clones from (d), in which, however,only one clone at most may originate from a particular group, andcarrying out sequencing reactions on these selected clones. Thepreparation of these clones can for example be achieved by atransformation of the ligated DNA into suitable host cells (preferablyE. coli cells) after the cloning step according to (d), an amplificationof the gene bank obtained in this manner and an examination ofindividual clones from this gene bank for the presence of an insertedDNA vector according to known methods (e.g. preparative gelelectrophoresis and isolation of plasmid DNA).

The sequencing reaction on the selected clones is in principle carriedout as in the already known multiplex sequencing method either by achemical method or by an enzymatic chain termination method. In thisprocess the individual base-specific reactions (either according to theSanger technique or according to the Maxam-Gilbert technique) arepreferably carried out directly on the mixture of different DNAfragments i.e. simultaneously.

In a chemical method according to Maxam-Gilbert, the DNA fragments to besequenced including the adaptors must firstly be cut out from thevector. This can for example be accomplished at the restriction sites atwhich the cloning into the vector has also taken place. In this mannerthe DNA fragments cloned in step (d) are released again. However,"rarely occurring" cleavage sites are preferably used for this purposewhich are in the vicinity of the cloning site, preferably at a distanceof less than 100 nucleotides. By this means the probability of acleavage within the DNA to be sequenced is extremely low. The cut outfragments are subsequently purified of vector DNA. This is preferablyachieved by preparative Agarose gel electrophoresis. In this way oneobtains a mixture of DNA fragments in which each DNA fragment hasadaptor molecules in the region of its two ends that are different fromthe adaptor molecules of another DNA fragment. This mixture issubsequently treated according to a known Maxam-Gilbert protocol. Thisgenerally involves a division of the mixture into four aliquots in whicheach aliquot is treated with a different, base-specific chemical reagentthat finally leads to a statistical cleavage of the DNA fragments at therespective bases. Consequently a population of sequencing products isproduced whose sequence can be determined in a next step.

In an enzymatic chain termination method according to Sanger it is notnecessary to cut out the DNA fragments from the vector. The mixture ofvectors with different cloned DNA fragments is firstly denatured. Aprimer oligonucleotide that is complementary with a region of the vectorin the vicinity of the cloned DNA fragment or/and with the adaptor andtherefore binds to the denatured DNA is added to the resultingsingle-stranded DNA. If it is intended to carry out the sequencing ofthe DNA fragment from two sides then two different primer molecules canbe added to each preparation. After dividing the mixture obtained inthis way into four aliquots a polymerisation reaction with a DNApolymerase is carried out according to the known Sanger protocol in eachcase in the presence of a different chain terminating molecule. Thepopulation of sequencing products produced in this manner issubsequently treated further as described in the following.

Step (f) of the method according to the invention, the analysis of thesequencing reaction using detectable hybridization probes that in eachcase are specific only for one single clone from the total number ofclones comprises generally the following steps:

(f1) separating the sequencing products obtained by the sequencingreactions from (e) according to their size in which correspondingsequencing products of a particular reaction from several clones areseparated together,

(f2) transferring the separated sequencing products onto a suitablecarrier for binding nucleic acids and if desired, immobilization of thetransferred sequencing products on the carrier,

(f3) reversible hybridization of the carrier with (i) a first detectablehybridization probe or (ii) with two or several selectively detectablehybridization probes in which each probe is in each case only specificfor a single clone from (e) and in which the specificity of each probeis defined by a hybridization to a particular target sequence carriedout under the reaction conditions, which is formed from a particularadaptor and if desired neighbouring vector sequences,

(f4) analysis of the results from (f3), removal of the boundhybridization probes from the carrier and

(f5) if desired, repetition of steps (f3) and (f4) with furtherdetectable hybridization probes which differ from the other probes inthat they bind to other target sequences.

The separation of the sequencing products (f1) is preferably carried outby gel electrophoresis, particularly preferably by a denaturingpolyacrylamide gel electrophoresis in a special sequence gel. A membranemade of nitrocellulose, nylon, polyvinylidene fluoride or chemicallyactivated cellulose (e.g. diazocellulose) is preferably used as thecarrier onto which the separated sequencing products are transferred(f2). The transfer of the DNA fragments onto the carrier and theirimmobilization is carried out with the aid of known blotting techniques(see e.g. Sambrook et al., A Laboratory Manual, Cold Spring HarborLaboratory Press, 1989).

This is followed by a reversible hybridization of the carrier with afirst detectable hybridization probe which in each case is only specificfor a single clone. It is, however, also possible to use two or morehybridization probes simultaneously provided that due to differentlabels they can be selectively detected concurrently. The specificity ofa probe is determined in this case solely by the group-specific orclone-specific adaptor. When using shorter adaptors, the hybridizationof the probe can take place not only at the adaptor itself but alsoadditionally at neighbouring vector sequences. The length of thehybridization region between the probe and the specific target sequenceis preferably 10 to 50 nucleotides, particularly preferably 15 to 40nucleotides. In this manner a hybridization is achieved that enables thedetermination of the nucleic acid sequence.

The removal of a probe hybridizing reversibly to the complementarytarget sequence from the carrier is also carried out according to knownmethods e.g. by heating above the melting point of the hybrid in thepresence of SDS (see e.g. Sambrook et al., supra).

Radioactively-labelled as well as non-radioactively-labelledhybridization probes can be used for the method according to theinvention. It is preferable to use non-radioactively-labelled probessuch as biotin, fluorescent, luminescent, digoxigenin or/and enzyme(e.g. peroxidase or alkaline phosphatase) labelled probes. It isparticularly preferable to use probes that are labelled with digoxigeninor derivatives thereof. Probes according to the invention can beproduced in a simple manner by chemical synthesis in which theincorporation of labelled nucleotides can be achieved during thesynthesis. On the other hand the probe can also be radioactivelylabelled subsequently e.g. by 5'-phosphorylation.

In the attached drawing a particularly preferred embodiment of themethod according to the invention for simultaneous sequencing is againshown schematically. The components required as starting material are avector (A) and two adaptor molecules (B) and (C) which are composed ofdouble-stranded DNA and each of which are provided on one side with ablunt end and on the other side with a protruding end.

FIG. 1 shows the starting materials.

The vector is hydrolysed with two different restriction enzymes each ofwhich produce protruding, non-compatible ends.

FIG. 2 shows this hydrolysis.

The sequencing DNA with blunt ends (D) is ligated to the adaptormolecules and cloned into the opened vector. For this the sequencing DNAobtained is preferably separated by Agarose gel electrophoresis and aregion of a desired size, preferably 800 bp to 1200 bp is cut out fromthe gel and eluted.

FIG. 3 illustrates this schematically.

The vector obtained in this way with inserted DNA can be proliferated bytransformation into suitable cells (in general E. coli cells) andisolated.

The present invention in addition concerns a method for converting analready existing gene bank into a modified gene bank on which asimultaneous sequencing of nucleic acids (as described above) can becarried out. The term "gene bank" means in this case that a number ofdifferent DNA fragments (to be sequenced) are present as insertions in abase vector. The method for modifying a gene bank is based on theintroduction of a double-stranded DNA adaptor molecule into the vectorin the vicinity of the insertions to be determined by which means whenseveral different adaptor molecules are used it is possible to dividethe gene bank into a number of groups on which a simultaneous sequencingcan be carried out.

This method comprises the steps:

(a) providing any desired gene bank consisting of a multitude ofdifferent DNA fragments which have been cloned into a base vector inwhich the base vector contains at least one singular, rarely occurringenzymatic cleavage site in the vicinity of the cloned DNA fragments,

(b) linearizing the gene bank by cleavage at a rarely occurring cleavagesite according to (a),

(c) dividing the gene bank into several groups,

(d) ligating the divided, linearized gene bank with a double-strandedDNA adaptor, the double-stranded region of the adaptor having a lengthof ≧5 nucleotides and ends on both sides which match the cleavage siteof the base vector, whereby an adaptor with a different sequence isligated to each group of the gene bank and

(e) if desired, separating the desired ligation products fromby-products.

In this process the nucleotide sequence of the adaptor molecule ispreferably selected so that the cleavage site in the vector iseliminated by introduction of the adaptor. This enables the desiredligation products to be separated from by-products by recleaving themwith the restriction enzyme used in step (b) for linearizing the genebank. In this manner a religated vector (without adaptor moleculeinsertion) is opened again so that the vector with cloned DNA isobtained as the only transformable product. The "rarely occurringcleavage site" in this method is as defined above i.e. it preferably hasa recognition sequence of at least 8 nucleotides length or/and has atleast once the nucleotide sequence 5'-CG-3', e.g. a NotI, SfiI, RsrII,SgrAI, SwaI, PacI, AscI, PmeI, Sse8387I, SrsI or I-SceI recognitionsite.

A particularly preferred embodiment for converting conventional genebanks into gene banks on which a simultaneous DNA sequencing is possibleis described schematically in the following. In this process one startswith a gene bank in a base vector (A) which has a rarely occurringrestriction site (e.g. NotI) in the vicinity of the insertion. Modernhigh performance vectors (e.g. pBsSk vectors) are preferably used asbase vectors. Furthermore one requires a set of special adaptoroligonucleotides (B) as starting material which have compatible ends forthe restriction site. The adaptor molecules are preferablydephosphorylated and the sequences next to the protruding regions at theend should preferably not correspond to the NotI recognition sequence.The double-stranded region of the adaptor molecule contains the targetsequences required for the simultaneous DNA sequencing (to which thehybridization probe is later to bind). Therefore a correspondingly highnumber of adaptor molecules with "different nucleotide sequence" (seeabove definition) is required. In particular no cross-hybridizationshould occur between the individual adaptor molecules which wouldinterfere with the sequence determination.

In a first step the vector is hydrolysed at a rarely occurringrestriction site (in this case NotI) in the vicinity (preferably ≦100nucleotide distance) of the DNA insertion and divided into severalgroups. Subsequently each group of the hydrolysed vector is ligated witha different, the double-stranded adaptor molecule.

In this process undesired by-products (2) and (3) are formed in additionto the desired product (1) of which (3) is non-transformable (andtherefore does not require a special separation) and (2) can be againlinearized by recleaving with NotI and thus made untransformable (whenthe nucleotide sequence of the adaptor molecule is selected so that theNotI site is removed). This is shown schematically in FIG. 4.

The sequencing reaction on such a gene bank is carried out according tothe principle which has already been described above.

In the modification of a gene bank according to the invention one can onthe one hand use a double-stranded adaptor molecule which has asymmetrical sequence and is composed of two identical self-complementaryoligonucleotides, but on the other also use an adaptor molecule whichhas an unsymmetrical sequence. The use of an unsymmetrical adaptormolecule is preferred. In this case a mixture of two hybridizationprobes (each of which is complementary to one strand of the adaptormolecule but are not self-complementary) must be used for the analysisof a sequence reaction.

If a gene bank in a base vector is used as the starting product for themethod which contains at least one singular, rarely occurringrestriction site on each of the two sides in the vicinity of the clonedDNA fragments to be sequenced, then steps (b) to (e) of the methodaccording to the invention can be carried out twice. In this way thereis then a specific adaptor molecule located on both sides of the clonedDNA fragment, each of which can bind a specific hybridization probe in asimultaneous sequencing process as described above so that a sequencingof the cloned DNA fragment starting at both ends of the fragment ispossible.

It is in addition intended to elucidate the invention by the followingsequence protocols and examples.

SEQ ID NO: 1 Adaptor (1)

SEQ ID NO: 2 Adaptor (2) In the sequence protocol the complementarystrand in the 5'-3' direction of the adaptor used is shown.

SEQ ID NO: 3 Adaptor (3)

SEQ ID NO: 4 Adaptor (4) In the sequence protocol the complementarystrand in the 5'-3' direction of the adaptor used is shown.

SEQ ID NO: 5 Multiplex vector 10--Plex 10E

SEQ ID NO: 6 Multiplex vector 10--Plex 10P

SEQ ID NO: 7 Multiplex vector 19--Plex 19E

SEQ ID NO: 8 Multiplex vector 19--Plex 19P

SEQ ID NO: 9 Adaptor GB1. The adaptor DNA is composed in the 5'-3'direction of the bases 1-23 and in the 3'-5' direction of the basescomplementary to 5-27.

SEQ ID NO: 10 Adaptor GB2. The adaptor DNA is composed in the 5'-3'direction of the bases 1-22 and in the 3'-5' direction of the basescomplementary to 5-26.

SEQ ID NO: 11 Oligonucleotide BG1A

SEQ ID NO: 12 Oligonucleotide BG1B

SEQ ID NO: 13 Oligonucleotide BG2A

SEQ ID NO: 14 Oligonucleotide BG2B

SEQ ID NO: 15 T3 sequencing primer

EXAMPLES Example 1

Constructing a gene bank according to the multiplex procedure

Fragmentation of the DNA:

10 μg chromosomal yeast-DNA (dissolved in 50 μl 50 mmol/l Tris HCl, 10mmol/l MgCl₂, 10 mmol/l DTT pH 7.8) was sonified in an ultrasonic bath(Sonorex RK255H, BANDELIN, frequency: 35 kHz; power: 160/320 W) untilthe fragments obtained were smaller than 5 kbp on analysis by means ofAgarose gel electrophoresis.

Polishing the ends and separation according to size:

In order to polish the ends, the DNA fragments were reacted for 30minutes in a volume of 40 μl with Klenow polymerase (0.25 U/μl) and T4DNA polymerase (0.15 U/μl) in the presence of dNTPs (0.025 mmol/l).After inactivation of these enzymes (10 min. heating at 70° C.), aportion of this DNA was applied to an Agarose gel (0.8% low meltingpoint Agarose, 1×TBE) and separated by electrophoresis (1 h at 4 V/cm).

Isolation of the ca. 1 kbp long fragments:

The region of the gel which contains fragments between 0.5 and 1.1 kbplength was cut out from the gel. The DNA fragments were eluted byfreezing the Agarose and subsequent centrifugation and precipitated byaddition of 1/10 volumes sodium acetate (3 mol/l, pH 7) and 0.8 volumesisopropanol. The precipitated DNA was washed with 70% EtOH (v/v), driedand taken up in TE (10 mmol/l Tris-HCl,0.1 mmol/l EDTA pH 8).

Cloning of the fragments in the multiplex vector 10 or 19

About 20 ng of the fragments obtained was ligated with the aid of T4 DNAligase (0.3 Weiss units/μl) in a volume of 20 μl (50 mmol/l Tris-HCl, 10mmol/l MgCl₂, 10 mmol/l DTT, pH 7.8, 0.5 mmol/l rATP) with 20 ng of themultiplex vector 10 or 19 (corresponding to vectors 1 or 3 of themultiplex kit from the Millipore Co.) (22 h at 16° C.). The multiplexvector was previously hydrolysed under standard conditions with SmaI andsubsequently dephosphorylated.

Transformation and culture of DH5a cells

Competent DH5α cells obtained commercially were transformed with a tenthof the ligation mixture according to the instructions of the BRL Companyand cultured while selecting for tetracycline. 5×10⁵ independentcolonies were obtained per μg DNA in relation to the vector used.

Constructing gene banks which contain exclusively plasmids with clonedyeast DNA

A LB plate with about 1000 of the colonies obtained is cultured at 37°C. until the colonies have reached a diameter of ca. 1 to 2 mm. Then thecolonies are suspended in LB medium with the aid of a glass spatula,removed from the plate and cultured in a volume of 3 ml while selectingfor tetracycline for a further 2 hours. Afterwards the bacteria aresedimented by centrifugation. The plasmid DNA is isolated by alkalinelysis of the cells under standard conditions and purified by columnchromatography (Qiagen, mini-preparation). A portion of this DNA (ca. 2μg) is applied to an Agarose gel (0.8% low melting point Agarose, 1×TBE,0.5 mg/ml ethidium bromide) and separated by electrophoresis (1 h at 4V/cm). The region of the gel between the "supercoiled form" and the"relaxed form" of the multiplex vector 10 or 19 contains the plasmid DNAwith cloned yeast DNA. This region of the gel is cut out. The DNAcontained therein is eluted by freezing the Agarose and subsequentcentrifugation and precipitated by addition of 1/10 volumes sodiumacetate (3 mol/l, pH 7) and 0.8 volumes isopropanol. The precipitatedDNA is washed with 70% EtOH (v/v), dried and taken up in TE (10 mmol/lTris-HCl, 0.1 mmol/l EDTA, pH 8). Competent DH5α cells are transformedwith a portion of the DNA obtained and cultured on LB plates whileselecting for tetracycline.

Example 2

Constructing a gene bank according to the described method

Chromosomal DNA of yeast was disintegrated into fragments which aresmaller than 5 kbp as described in example 1 by sonication withultrasound. The ends of these DNA fragments were polished by treatmentwith Klenow polymerase and T4 DNA polymerase. After inactivation ofthese enzymes (10 min. at 70° C.), 2 μg of this DNA was reacted in avolume of 20 μl (50 mmol/l Tris-HCl, 10 mmol/l MgCl₂, 10 mmol/l DTT pH7.8, 1 mmol/l rATP) with T4 DNA ligase (50 Weiss units per ml) and 2 μgof each of the following adaptor molecules:

Adaptors

    (1):5'-AATTCCATAACTGTAACCTTTAAC-3' EcoN(24-mer)            SEQ ID NO:1

    (2): 3'-GGTATTGACATTGGAAATTG-5' EcoH(20-mer)               SEQ ID NO:2

    (3): 5'-CTATTTGTAATTCCGCTGCA-3' PstH(20-mer)               SEQ ID NO:3

    (4): 3'-GATAAACATTAAGGCG-5' PstN(16-mer)                   SEQ ID NO:4

The processing of the DNA fragments obtained in this way is carried outas described in example 1: After the ligation the DNA fragments wereapplied to a 0.8% Agarose gel and separated. The fragments which werebetween 0.5 and 1.1 kbp long were eluted and purified. Afterwards theywere cloned into the multiplex vector 10 or 19 which in contrast toexample 1 had stepped non-mutually compatible ends by previous treatmentwith EcoRI and PstI. Competent DH5α cells were transformed with theligation mixture and cultured on LB plates while selecting fortetracycline.

3×10⁶ independent colonies per μg DNA were obtained relative to thevector used and thus about 6 times more colonies than in example 1.

Gene banks which contained exclusively plasmids with cloned yeast DNAwere constructed as described in example 1.

Example 3

Simultaneous sequencing of gene banks which were constructed accordingto the multiplex procedure or according to the new procedure.

Single colonies were set up by streaking gene banks which were obtainedaccording to example 1 and example 2. 1 colony from each of the two genebanks were combined in 10 ml LB medium and suspended. The medium wasshaken at 37° C. until the suspension had an optical density of 2 at awavelength of 600 nm. Afterwards the bacteria were sedimented bycentrifugation. The plasmid DNA was isolated by alkaline lysis of thecells under standard conditions and purified by column chromatography(Qiagen, minipreparations).

The mixture of the two plasmid DNAs (8 μg) was sequenced using amodified T7 polymerase (sequenase) and the protocols of the USB Co. Amixture of the oligonucleotides Plex E and Plex P (in each case 40 ngper mixture) was used as the sequencing primer which was used as astandard for sequencing according to the multiplex procedure. Thesequencing products obtained were separated on a sequence gel (40 cmlong, 0.4 mm thick, 6% polyacrylamide, 7 mol/l urea; buffer: 1×TBE),transferred onto a neutral nylon membrane by a capillary blot andimmobilized on the membrane by irradiating with 256 nm UV light. Thesequence products were detected by hybridization withdigoxigenin-treated oligonucleotides which had been obtained by reactingthe unmodified oligonucleotides with terminal deoxynucleotidyltransferase and digoxigenin-treated dUTP (Boehringer Mannheim). Thecorresponding sequence ladders were made visible with the aid of thedigoxigenin detection system of the Boehringer Mannheim Company (Fabfragments of anti-digoxigenin antibodies) and subsequentchemiluminescence. After the detection of a sequence reaction, thehybridized oligonucleotide was removed by heating the membrane understandard conditions. The membrane was in each case reacted once with oneof the following digoxigenin-treated oligonucleotides:

Multiplex vector 10

    Plex 10E (5'-TATATATAGGGTATTAGGTG 3')                      SEQ ID NO: 5

    Plex 10P (5'-TGAGTATATTGATGATTAGG 3')                      SEQ ID NO: 6

Multiplex vector 19

    Plex 19E (5'-AGAAGTTAATGTAGGGTTGG3')                       SEQ ID NO: 7

    Plex 19P (5'-GTGATAAGTAGAGTTGGTTG-3'),                     SEQ ID NO: 8

PstH, EcoH (see example 2).

Result:

In each of the hybridizations carried out, an independent unequivocalsequence could be determined.

Example 4

Conversion of an already existing gene bank into a gene bank which issuitable for simultaneous sequencing.

Isolation of the plasmids containing cDNA:

A cDNA bank from human heart set up in the EcoRI cleavage site of thephage vector lambda ZAP II and commercially available from theStratagene Co. (San Diego, U.S.A.) (Catalogue No. 936208) was firstlyconverted into the double-stranded phagemid form according to theinstructions of the manufacturer.

Hydrolysis with NotI and attachment of the adaptors:

1 μg of the total DNA isolated from the phagemid bank was cleaved understandard conditions with the enzyme NotI. In two separated reactions,300 ng of the adaptor DNA (mixture 1: adaptor GB1; mixture 2: adaptorGB2) was added in each case to 500 ng of the cleaved DNA. The adaptorswere ligated with the DNA (10 h; 12° C.) by reaction with T4 DNA ligase(50 Weiss units/ml) in a volume of 20 μl (50 mM Tris-HCl, 10 mM MgCl₂,10 mM DTT, pH 7.8, 1 mM ATP). The adaptors used have the followingsequence:

GB1:

    5'-GGCCACATAACTCAAATCTCAAA.sub.- 3'                        SEQ ID NO: 9

    3'-TGTATTGAGTTTAGAGTTTCCGG-5'                              SEQ ID NO: 16

GB2:

    5'-GGCCAGCTATCTCGTAATTGCT-3'                               SEQ ID NO: 10

    3'-TCGATAGAGCATTAACGACCGG-5'                               SEQ ID NO: 17

Separation of the excess adaptors

After the ligation the reaction mixture is heated for 10 minutes to 65°C. and subsequently cooled on ice. The reaction mixtures are applied toan Agarose gel (0.8% low melting point Agarose, 1×TBE, 0.5 mg/mlethidium bromide) and separated by electrophoresis (1 h at 4 V/cm). Theregion of the gel containing plasmid DNA (larger than linearized pBsSKIIDNA) is cut out. The DNA contained therein is eluted by freezing theAgarose and subsequent centrifugation and precipitated by addition of1/10 volumes sodium acetate (3 mol/l, pH 7) and 0.8 volumes isopropanol.The precipitated DNA is washed with 70% EtOH (v/v), dried and taken upin TE (10 mmol/l Tris-HCl, 0.1 mmol/l EDTA, pH 8).

Circularization of the plasmid DNA with adaptors at both ends byannealing the cohesive ends.

Ca. 50 ng of the isolated DNA is firstly stored for 20 minutes at 65° C.and then for a further 2 h at 37° C. in a volume of 20 μl (50 mMTris-HCl, 10 mM MgCl₂, 10 mM DTT, pH 7.8).

Construction of gene banks

Competent DH5α cells obtained commercially were transformed with a tenthof the ligation mixtures according to the instructions of the BRL Co.The bacteria are streaked out on LB plates and cultured while selectingfor ampicillin. The gene banks constructed in this manner contained--inrelation to the vector used--1 to 6×10⁶ independent colonies per μg ofDNA used.

In each case one colony from each of the two gene banks was removed andcultured together in 10 ml LB medium. Plasmid DNA was isolated asdescribed in example 3 and the mixture of the two plasmid DNAs (8 μg)was sequenced using a modified T7 polymerase (sequenase) and theprotocols of the USB Co. The T3 sequencing primer(5'-ATTAACCCTCACTAAAG-3'(SEQ ID NO: 15), in each case 40 ng per mixture)was used as the sequencing primer. The sequence products obtained wereseparated on a sequence gel (40 cm long, 0.4 mm thick, 6%polyacrylamide, 7 mol/l urea; buffer: 1×TBE), transferred onto a neutralnylon membrane by a capillary blot and immobilized on the membrane byirradiation with 256 nm UV light. The sequence products were detected byhybridization with digoxigenin-treated oligonucleotides which have beenobtained by reaction of unmodified oligonucleotides with terminaldeoxynucleotidyl transferase and digoxigenin-treated dUTP (BoehringerMannheim). The corresponding sequence ladders are made visible with theaid of the digoxigenin detection system of the Boehringer Mannheim Co.(Fab fragments of anti-digoxigenin antibodies) and subsequentchemiluminescence. After the detection of a sequence reaction, thehybridized oligonucleotide was removed by heating the membrane understandard conditions.

The membrane is reacted in each case once with a mixture of theoligonucleotides BG1A and BG1B and subsequently with a mixture of theoligonucleotide BG2A and BG2B:

    BG1A: 5'-GAGTTATGTGGCCGCCACC-3'                            (SEQ ID NO: 11)

    BG1B: 5'-AGACGGCCACATAACTCAAA-3'                           (SEQ ID NO: 12)

    BG2A: 5'-CGAGATAGCTGGCCGCCA-3'                             (SEQ ID NO: 13)

    BG2B: 5'-AGAGCGGCCAGCTATCTC-3'                             (SEQ ID NO: 14)

Result:

In each of the hybridizations carried out it was possible to determinean independent unequivocal sequence.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 17                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       AATTCCATAACTGTAACCTTTAAC24                                                    (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       GTTAAAGGTTACAGTTATGG20                                                        (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       CTATTTGTAATTCCGCTGCA20                                                        (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 16 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       GCGGAATTACAAATAG16                                                            (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       TATATATAGGGTATTAGGTG20                                                        (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       TGAGTATATTGATGATTAGG20                                                        (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       AGAAGTTAATGTAGGGTTGG20                                                        (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       GTGATAAGTAGAGTTGGTTG20                                                        (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       GGCCACATAACTCAAATCTCAAA23                                                     (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      GGCCAGCTATCTCGTAATTGCT22                                                      (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      GAGTTATGTGGCCGCCACC19                                                         (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      AGACGGCCACATAACTCAAA20                                                        (2) INFORMATION FOR SEQ ID NO:13:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                      CGAGATAGCTGGCCGCCA18                                                          (2) INFORMATION FOR SEQ ID NO:14:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                      AGAGCGGCCAGCTATCTC18                                                          (2) INFORMATION FOR SEQ ID NO:15:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                      ATTAACCCTCACTAAAG17                                                           (2) INFORMATION FOR SEQ ID NO:16:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                      GGCCTTTGAGATTTGAGTTATGT23                                                     (2) INFORMATION FOR SEQ ID NO:17:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                      GGCCAGCAATTACGAGATAGCT22                                                      __________________________________________________________________________

We claim:
 1. A method for the simultaneous sequencing of nucleic acids,comprising:(a) preparing numerous DNA fragments whose sequences are tobe determined; (b) dividing the DNA fragments to be sequenced intoseveral groups; (c) ligating double-stranded DNA adaptors to both endsof the divided DNA fragments from step (b) to produce DNAfragment/adaptor complexes, wherein the adaptors have a double-strandedregion with a length of at least 5 nucleotides, have a first end that iscompatible to the ends of the DNA fragments and a second end suitablefor ligating into a vector, and wherein adaptors having differentnucleotide sequences are ligated to the DNA fragments of each of theseveral groups; (d) ligating DNA fragment/adaptor complexes from each ofthe several groups from step (c) into a base vector comprising aplurality of cleavage sites for a plurality of restrictionendonucleases, which base vector has been cleaved with at least one ofthe restriction endonucleases, wherein the cleavage produces endsmatching with the second end of one of the adaptors; (e) selecting onevector from each of the several groups produced in step (d) and carryingout sequencing reactions on each of the ligated DNA fragment/adaptorcomplexes present in the selected vectors to produce sequencingproducts; and (f) producing nucleotide sequence information byseparating the sequencing products of step (e) from one another andthereafter identifying the separated sequencing products by hybridizingthe separated sequencing products with at least one hybridization probewhich comprises a DNA sequence which is complementary or identical to a5 nucleotide portion of one adaptor of one single DNA fragment/adaptorcomplex.
 2. The method as claimed in claim 1, wherein the at least onehybridization probe comprises a DNA sequence which is complementary tothe DNA adaptor portion of the one single DNA fragment/adaptor complex.3. The method as claimed in claim 1, wherein the DNA fragments of step(a) have blunt ends.
 4. The method as claimed in claim 1, wherein theDNA fragments of step (a) have stepped ends.
 5. The method as claimed inclaim 1, wherein the second end of the double-stranded DNA adapters isstepped.
 6. The method as claimed in claim 1, wherein the length of thedouble-stranded region of the double-stranded DNA adapters is 10 to 40nucleotides.
 7. The method as claimed in claim 1, wherein thedouble-stranded DNA adapters ligated to each end of the divided DNAfragments consist of the same nucleotide sequence.
 8. The method asclaimed in claim 1, wherein the double-stranded DNA adapters ligated toeach end of the divided DNA fragments consist of different nucleotidesequences.
 9. The method as claimed in claim 8, wherein the second endsof the double-stranded DNA adapters are stepped.
 10. The method asclaimed in claim 9, wherein the stepped second ends of thedouble-stranded DNA adapters ligated to each end of the divided DNAfragments are non-complementary.
 11. The method as claimed in claim 1,wherein the base vector comprises an insertion region for inserting aDNA fragment/adaptor complex, the insertion region flanked on each sideby at least one restriction endonuclease recognition sequence whichcontains at least one of (1) a sequence of at least 7 nucleotides and(2) a 5'-CG-3' nucleotide sequence.
 12. The method as claimed in claim11, wherein the at least one restriction endonuclease recognitionsequence is selected from the group consisting of NotI, SfiI, RsrII,SgrAI, SwaI, PacI, AscI, PmeI, Sse83871, SrsI and I-SceI.
 13. The methodas claimed in claim 1, wherein each of the plurality of cleavage sitesis flanked by transcription terminators.
 14. The method as claimed inclaim 1, wherein the at least one hybridization probes isnon-radioactively labelled.
 15. The method as claimed in claim 14,wherein the at least one hybridization probe is non-radioactivelylabelled with a label independently selected from the group consistingof biotin, digoxigenin, a fluorescent label, a luminescent label, and anenzyme.
 16. The method as claimed in claim 1, wherein step (f) comprisesthe following steps:(f1) separating the sequencing products of step (e)according to the size thereof; (f2) transferring the separatedsequencing products from step (f1) onto a suitable carrier for bindingnucleic acids; (f3) reversibly hybridizing the separated sequencingproducts from step (f2) with at least one hybridization probe whichcomprises a DNA sequence which is complementary to one single DNAfragment/adaptor complex; and (f4) producing nucleotide sequenceinformation by analyzing the hybridization of the separated sequencingproducts from step (f3).
 17. The method as claimed in claim 16, whereinthe at least one hybridization probe comprises a DNA sequence which iscomplementary to the DNA adaptor portion of the one single DNAfragment/adaptor complex.
 18. The method as claimed in claim 16, whereinstep (f2) further comprises immobilizing the transferred separatedsequencing products on the carrier.
 19. The method as claimed in claim16, further comprising, after step (f4), removing the hybridized atleast one hybridization probe from the carrier, and thereafter repeatingsteps (f3) and (f4) using a different hybridization probe whichcomprises a DNA sequence which is complementary to a different singleDNA fragment/adaptor complex.
 20. A method for converting an alreadyexisting gene bank into a modified gene bank on which a simultaneoussequencing of nucleic acids can be carried out, wherein the alreadyexisting gene bank comprises a plurality of different DNA fragmentswhich have been ligated into a base vector at an insertion regionthereof, wherein the insertion region is flanked on a side by at leastone restriction endonuclease recognition sequence which contains atleast one of (1) a sequence of at least 7 nucleotides and (2) a 5'-CG-3'nucleotide sequence, the method comprising:(a) cleaving the gene bankwith an enzyme at the at least one restriction endonuclease recognitionsequence to produce numerous DNA fragments; (b) dividing the DNAfragments into several groups; and (c) ligating the divided DNAfragments from step (b) with a double-stranded DNA adaptor to produceligation products, wherein the adaptor has a double-stranded region witha length of at least 5 nucleotides and ends on both sides which aresuitable for ligation with the at least one restriction endonucleaserecognition sequence, and wherein an adaptor having a differentnucleotide sequence is ligated to a DNA fragment from each of theseveral groups.
 21. The method as claimed in claim 20, furthercomprising, after step (c),(d) separating the ligation products fromby-products.
 22. The method as claimed in claim 20, wherein thedouble-stranded DNA adaptor has a sequence which is selected such thatthe at least one restriction endonuclease recognition sequence iseliminated by ligation of the double-stranded DNA adaptor in step (c).23. The method as claimed in claim 21, wherein step (d) comprisesrecleaving the ligation products with the enzyme used in step (a). 24.The method as claimed in claim 20, wherein the at least one restrictionendonuclease recognition sequence is selected from the group consistingof NotI, SfiI, RsrII, SgrAI, SwaI, PacI, AscI, PmeI, Sse83871, SrsI andIsceI.
 25. The method as claimed in claim 20, wherein the adaptormolecule has a symmetrical sequence.
 26. The method as claimed in claim20, wherein the adaptor molecule has an unsymmetrical sequence.
 27. Themethod as claimed in claim 20, wherein the insertion region is flankedon each side by different restriction endonuclease recognition sequenceseach containing at least one of (1) a sequence of at least 7 nucleotidesand (2) a 5'-CG-3' nucleotide sequence, and wherein the steps (a)-(c)are carried out for each at least one restriction endonucleaserecognition sequence.